As described above, this project has three main goals. 1. Analysis of startle modulation. We previously demonstrated that intense acoustic stimuli elicit two types of startle response in zebrafish larvae: rapid short latency responses and lower performance long latency responses. All fish can generate both types of response, but which response emerges is unpredictable from trial to trial. We aim to understand how fish select to deploy a short or long latency response. Short latency responses are modulated in a similar fashion to startle responses in mammals where startle magnitude is inhibited when the startle stimulus is preceded by a weak auditory prepulse. This form of startle modulation, termed prepulse inhibition, is diminished in several neurological conditions including schizophrenia. Previously, we conducted a screen to identify fish carrying genetic mutations resulting in a reduction in prepulse inhibition. We are now performing linkage analysis using these fish to map the genetic mutations in the mutants and identify genes required for prepulse inhibition. In parallel, we are analyzing how long latency responses are generated. Brainstem neurons which trigger a motor response must belong to the restricted cohort of neurons which project to the spinal cord. We are therefore sequentially ablating neurons of this class using a pulsed nitrogen laser, then probing the stimulus threshold and magnitude of the long latency startle response system. Together, these approaches will allow us to find neuronal mechanisms for the implementation of behavioral choice in zebrafish larvae. 2. Functional mapping of serotonergic neuronal architecture. We have recently found that the startle response is sensitized when larvae swim away from negative stimulus environments. This is consistent with findings that the startle response is modified in higher vertebrates by anxiety, a state regulated by serotonergic signaling. We hypothesized that the serotonergic system may therefore also regulate the startle response in larval zebrafish. As a first step towards studying this phenomenon we have generated transgenic fish expressing the GAL4 transcription factor in serotonergic neurons. This enables us to genetically manipulate these neurons by crossing the fish to lines carrying reporter genes under the control of the UAS promoter, for example to a UAS:Nitroreductase line to genetically ablate neurons. Consistent with our hypothesis, nitroreductase ablation of serotonergic neurons alters startle responsiveness. We are now using two-photon based laser ablation to destroy single serotonergic neurons in vivo to identify the subset of neurons which modulate startle responses. As a second approach to identifying cells which modulate startle, we are generating transgenic fish in which the UAS promoter drives expression of mEOS-FP, a monomeric photoconvertible fluorescent protein. This system will allow us to trace the connections of single serotonergic neurons in vivo, and identify those which project to startle response circuitry. We are now extending our analysis of the role of serotonergic neurons by probing fish with serotonergic lesions in a battery of behavioral tests. Ultimately this will allow us to establish a neuronal level functional map of serotonergic anatomy. 3. Development of new tools for analysis of neural circuits involved in motor behavior. The relatively simple nervous system of zebrafish larvae and restricted range of motor behaviors opens up the possibility of identifying neuronal pathways which underlie the entire behavioral repertoire. For this to be feasible, it would be extremely useful to have reporter lines which would enable the manipulation of small groups of neurons known to be involved in a particular motor behavior. We are therefore performing an enhancer trap screen using a GAL4 reporter vector to identify lines with restricted patterns of neuronal expression. These lines will be used to genetically ablate trapped neurons and larvae screened for defects in locomotor behavior. We have established an automated screening platform which allows us to rapidly screen the entire repertoire of locomotor behaviors. Computational analysis of responses gives us a robust and sensitive measure of performance. This screen will thus generate a set of reporter lines which identify and provide experimental access to cohorts of neurons linked to specific behaviors. These lines will constitute a unique resource for decoding the developmental genetics and anatomical basis of behavior in zebrafish larvae. This is the first time such a screen has been attempted in a vertebrate organism.